The cloning of the gene encoding the KlGpa1p subunit was achieved by standard PCR techniques and by screening a Kluyveromyces lactis genomic library using the PCR product as a probe. The full-length open reading frame spans 1,344 nucleotides including the stop codon, The deduced primary structure of the protein (447 amino acid residues) strongly resembles that of Gpa1p, the G-protein alpha subunit from Saccharomyces cerevisiae involved in the mating pheromone response pathway. Nevertheless, unlike disruption of Gpa1 from S. cerevisiae, disruption of KlGpa1 rendered viable cells with a reduced capacity to mate. Expression of a plasmidic KlGpa1 copy in a Delta Klgpa1 mutant restores full mating competence; hence we conclude that KlGpa1p plays a positive role in the mating pathway, Overexpression of the constitutive subunit K1Gpalp(K-364) (GTP bound) does not induce constitutive mating; instead it partially blocks wild-type mating and is unable to reverse the sterile phenotype of Delta Klgpa1 mutant cells. K. lactis expresses a second G alpha subunit, KlGpa2p, which is involved in regulating cyclic AMP levels upon glucose stimulation. This subunit does not rescue Delta Klgpa1 cells from sterility; instead, overproduction of KlGpa2p slightly reduces the mating of wild-type cells, suggesting cross talk within the pheromone response pathway mediated by K1Gpalp and glucose metabolism mediated by KlGpa2p, The Delta Klgpa1 Delta Klgpa2 double mutant, although viable, showed the mating deficiency observed in the single Delta Klgpa1 mutant.
Última actualización: 06/12/2021